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  • Title: Estradiol attenuates mitochondrial depolarization in polyol-stressed lens epithelial cells.
    Author: Flynn JM, Cammarata PR.
    Journal: Mol Vis; 2006 Apr 04; 12():271-82. PubMed ID: 16617294.
    Abstract:
    PURPOSE: This study examined the state of mitochondrial physiology subsequent to exposing lens epithelium to high ambient galactose (Gal), which upon conversion to galactitol (GalOH) and resultant intracellular accumulation thereof, leads to profound destabilization of mitochondrial membrane potential (Deltapsim). Further, we determined whether the aldose reductase (AR) inhibitor, Sorbinil, or estrogen (17beta-E2, and its isomer, 17alpha-E2, which exhibits marginal binding affinity for estrogen receptor), administered prior to and concomitant with Gal exposure might prevent or delay mitochondrial membrane depolarization. METHODS: Secondary cultures of bovine lens epithelial cells (BLECs), as well as a virally-transformed human lens epithelial cell line (HLE-B3), were maintained in 40 mM galactose (Gal) for up to seven days in the presence and absence of Sorbinil, 17beta-E2 or 17alpha-E2. Endogenous accumulation of reactive oxygen species (ROS) was assessed by loading cells with H2DCF-DA, which upon oxidation in the presence of ROS transitions to the fluorescent compound, DCF. To assess Deltapsim, confocal microscopy was employed in conjunction with the potentiometric dye, JC-1. Intracellular polyol content was determined by gas chromatography. Cells were monitored for apoptosis and necrosis as determined by annexin V-propidium iodide staining and visualized by confocal fluorescence microscopy. RESULTS: BLECs, more so than HLE-B3 cells, accumulate high intracellular levels of GalOH upon exposure to high ambient Gal. BLECs were significantly depolarized while HLE-B3 cells showed little depolarization over the same course of Gal exposure. The addition of either 17alpha-E2 or 17beta-E2 to BLECs, over a dose range of 0.01 microM to 1.0 microM, prevented mitochondrial membrane depolarization as did the addition of 0.1 mM Sorbinil. The polyol content in BLECs after 3 days of exposure to Gal was 282 nmol/mg protein. Co-addition of Sorbinil during the 3-day exposure period prevented any significant accumulation of GalOH. Co-administration of either isoform of estrogen did not block GalOH synthesis and the level of attained intracellular accumulation was similar to that of Gal alone. The observed accumulation of ROS from HLE-B3 cells subsequent to 3 days of Gal exposure was negligible and consistent with that of control cells maintained in physiological medium. Intracellular accumulation of ROS with 3-day, Gal-maintained BLECs, exhibited a marginal but statistically significant increase over control cells maintained in physiological medium (5.5 mM glucose) and similar levels of ROS were generated irrespective of the presence of estrogen with Gal. Bolus addition of 100 microM hydrogen peroxide to 3-day, Gal plus Sorbinil-maintained BLECs failed to induce a change in mitochondrial membrane potential. Evidence of apoptosis or necrosis was negligible through 7 days of sustained exposure to high ambient Gal. CONCLUSIONS: Polyol accumulation promotes mitochondrial membrane depolarization and the decrease in Deltapsim is prevented by prior addition and co-administration of Sorbinil or estrogen with Gal. Unlike Sorbinil, estrogens' mode of action is not via the inhibition of aldose reductase activity. The data supports the theory that with Gal plus estradiol-treated cells, at a given intracellular polyol load, a larger portion of the mitochondrial population retains Deltapsim, and hence continues to function relative to Gal-treated cells. Results with 17alpha-E2 indicate that maintaining Deltapsim, in the face of chronic polyol accumulation, is likely to be mediated via a nuclear estrogen receptor-independent mechanism. The failure of supraphysiological levels of hydrogen peroxide added to Gal plus Sorbinil-maintained BLECs to depolarize mitochondria indicates that polyol accumulation, not ROS generation, is the causative factor responsible for the loss of mitochondrial membrane potential.
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